Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation

This information is current as Peishan Lee, Zilu Zhu, Janna Hachmann, Takuya Nojima, of September 24, 2021. Daisuke Kitamura, Guy Salvesen and Robert C. Rickert J Immunol published online 28 December 2016 http://www.jimmunol.org/content/early/2016/12/28/jimmun ol.1502518 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 28, 2016, doi:10.4049/jimmunol.1502518 The Journal of Immunology

Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation

Peishan Lee,*,† Zilu Zhu,* Janna Hachmann,*,‡ Takuya Nojima,x Daisuke Kitamura,x Guy Salvesen,* and Robert C. Rickert*

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-kB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-kB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling

where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of Downloaded from MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity. The Journal of Immunology, 2017, 198: 000–000. http://www.jimmunol.org/ ctivation of NF-kB has emerged as one of the most mice exhibit a reduction in the marginal zone (MZ) and B1 B cell crucial steps in mounting an effective immune response, subsets, decreased serum IgM and IgG3 levels, as well as impaired A regulating a wide array of genes essential for immune Ab responses to both T dependent (TD) and T independent (TI) Ags cell survival and function. NF-kB signaling can occur via the ca- (5, 6). A subsequent study unraveled a new role for MALT1 in nonical route, in which the major players are NF-kB dimers com- mediating BAFF-induced non-canonical NF-kB signaling specifically posed of RelA, c-Rel, and p50, or via the alternative non-canonical in MZ B cells, where MALT1 deficiency resulted in diminished route that is mediated by the Relb and p52 heterodimers (1). The BAFF-induced p100 processing and cell survival (7). Although the canonical NF-kB pathway becomes transiently activated after Ag detailed mechanism has not been completely elucidated, the defect in receptor engagement via the assembly of a signaling platform MALT1-deficient B cells has been associated with nuclear translo- by guest on September 24, 2021 composed of scaffold proteins CARMA-1 and BCL-10, and the cation of c-Rel upon triggering of the BCR (8). paracaspase MALT1 (termed the CBM complex), which relays Germinal centers (GCs) are specialized microenvironments signals from proximal kinases and adaptors to the core IkBkinase within the secondary lymphoid tissues that are formed at the complex (2, 3). Gene-targeting studies have revealed that mice height of an ongoing immune response and are critical for the deficient in any of the components of the CBM complex show optimization of TD humoral immune responses. Ag-activated defective B cell and T cell activation, resulting in an inadequate B cells entering the GC proliferate rapidly and undergo so- adaptive immune response (4). In the B cell compartment, Malt12/2 matic hypermutation and affinity maturation, ultimately leading to the generation of memory B and plasma cells producing high- affinity Abs (9). This process requires intricate interactions *Tumor Microenvironment and Cancer Immunology Program, Sanford Burnham among Ag-specific B and T cells, and follicular (FO) dendritic Prebys Medical Discovery Institute, La Jolla, CA 92037; †Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, CA 92037; cells (10). The initiation of the GC reaction is also dependent on ‡Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Dis- x a multitude of B cell-intrinsic factors modulating the BCR signal covery Institute, La Jolla, CA 92037; and Division of Molecular Biology, Research and, notably, deletion or mutation of NF-kBfamilymembers Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba 278- 0022, Japan often abrogates GC formation (11). For example, c-Rel–deficient ORCIDs: 0000-0002-4519-9151 (T.N.); 0000-0002-7933-6732 (G.S.). mice exhibit a severe impairment in GC formation upon im- Received for publication December 1, 2015. Accepted for publication November 28, munization, due to its critical role in regulating B cell survival 2016. and cell cycle progression (12, 13). Also, it has recently been This work was supported by National Institutes of Health Grants R01AI41649 (R.C.R.), shown that c-Rel is essential for the maintenance of GC B cells R01GM099040 (G.S.), and F31CA165782 (P.L.). via activation of a metabolic program that promotes cell growth 2 2 Address correspondence and reprint requests to Prof. Robert C. Rickert, Sanford (14). Histologic staining of the spleen in Malt1 / mice im- Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Road, La Jolla, munized with TD Ag revealed a complete absence of GC for- CA 92037. E-mail address: [email protected] mation (5). Furthermore, MALT1-deficient animals are devoid of The online version of this article contains supplemental material. spontaneously formed GC B cells in the Peyer’s patches, which Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ABC-DLBCL, activated B cell-like diffuse large B cell lymphoma; BM, bone marrow; Btk, Bruton’s are chronically exposed to Ags because of their anatomical lo- tyrosine kinase; FO, follicular; GC, germinal center; iGB, induced GC B cell; KO, cation (15). The lack of GC B cells also correlates with a severe knockout; MZ, marginal zone; PKC, protein kinase C; PNA, peanut agglutinin; TD, reduction in T FO helper (TFH) cells (15), because it has been T dependent; TFH,, T FO helper; TI, T independent; WT, wild type. shown that GC B cells are required to sustain the TFH pheno- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 type (16).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502518 2 B CELL-INTRINSIC MALT1 FUNCTION

For the initiation and progression of the GC response, T cell help of the serum samples, washed and detected with fluorescent conjugated is crucial. T cells can participate through the engagement of CD40 Abs against either IgM (clone II/41; eBioscience) and IgG1 (cloned on B cells and via signals delivered through costimulatory mole- A85.1; BD Biosciences). Mean fluorescence intensities of the SRBC- bound aIgM and aIgG1 Abs were plotted against the dilution factors, cules and cytokines (17, 18). It has been consistently reported that 2 2 and the values in the linear range were used for comparing the relative Malt1 / T cells exhibit impairment in activation, proliferation, Ab titers. and IL-2 production as well as NF-kB activation in response to Adoptive transfers TCR ligation (5, 6). However, it remains unclear whether the in- 2/2 ability of the Malt12/2 animals to mount GC reactions can be Splenic B cells were isolated from CD45.1 or Malt1 animals by CD43 3 6 entirely attributed to the defective T cell response. Moreover, the depletion and mixed at a 1:1 ratio. Then 1–2 10 B cells were transferred into the mMT mice via the tail vein. After 24 h, the mMT mice were role of MALT1 in B cell activation remains controversial with immunized with SRBCs and analyzed on day 7 postimmunization. respect to the requirement of MALT1 in BCR-stimulated proliferation and NF-kB activation (5, 6). Moreover, despite the Flow cytometry 2 2 established link to c-Rel translocation, Malt1 / animals do not Single-cell suspensions prepared from spleens and Peyer’s patches were fully phenocopy mice with c-Rel deletion, suggesting that MALT1 blocked with anti-CD16/32 (clone 2.4G2; BD Biosciences) and stained may regulate additional mechanisms in B cells. with the indicated combination of conjugated Abs for 30 min on ice. Live cells were assessed by forward and side scatter profiles. All cells were Loss-of-function mutations in MALT1 have also been discov- acquired on a FACSCanto flow cytometer using the FACSDiva software ered in human patients with combined immunodeficiency disor- (BD Biosciences) and data were analyzed using FlowJo software (Tree ders, resulting in abnormal T cell proliferation and failure to Star). The following Abs were obtained from eBioscience: anti-B220 activate NF-kB following treatment with the direct protein kinase (RA3-6B2), -CD3e (145-2C11), -CD21/35 (8D9), -CD23 (B3B4), Downloaded from C (PKC) activator PMA (19–21). Yet, the contribution of B cells -CD45.1 (A20), -CD4 (RM4-5), -IgD (11–26), -CD86 (PO3.1), -PD1 (J43), -ICOS (7E.17G9), -IgM (II/41), -CD5 (53.753), -CD62L (MZL-14), to the disease manifestations in these patients is still unclear. In -CD86 (PO3.1), -MHC class II (M5/114.15.2), -CD25 (PC61.5), and contrast, deregulated expression of MALT1 has been implicated in -CD69 (H1.2F3). Anti-GL7, -FAS (JO2), -CD45.2 (104), -CD138 (281-2), B cell lymphomagenesis, and a recent study demonstrated the -IgG1 (A85.1), and -CD80 (16-10A1) Abs were purchased from BD oncogenic potential of MALT1 in driving B cell lymphopoiesis Biosciences.

(22). Consequently, understanding the role of MALT1 in B cells is In vitro proliferation, cell cycle, and apoptosis assays http://www.jimmunol.org/ crucial from a clinical perspective, for the management of diseases B cells were isolated from splenocytes by negative magnetic-based including immunodeficiency and lymphoma. sorting of cells labeled with CD43 microbeads (Miltenyi Biotec). For To our knowledge, in this study we provide the first evidence the proliferation assay, purified B cells were labeled with eFluor670 that MALT1 is required in B cells for the GC response. MALT1 is (eBioscience) according to the manufacturer’s protocol and cultured in 6 critical for efficient proliferation and survival in response to BCR 96-well plates at a density of 10 cells/ml in complete RPMI 1640 (Cellgro; Corning) supplemented with 10% FBS (Sigma), 13 Penicillin/ signaling. Consequently, initiation of the GC response upon im- Streptomycin (Cellgro; Corning), 2 mM GlutaGro (Cellgro; Corning), munization is impaired in the absence of MALT1. We also show 13 MEM non-essential amino acids (Cellgro; Corning), 1 mM sodium that T cell-derived help fails to rescue the proliferation and sur- pyruvate (Cellgro; Corning), and 50 mM b-mercaptoethanol (Life vival defects in MALT1-deficient B cells. Lastly, we demonstrate Technologies), with or without various stimuli. The following stimuli by guest on September 24, 2021 that ectopic expression of Bcl-2 in MALT1-deficient B cells can were used at the indicated concentrations: 25 ng/ml recombinant murine BAFF (R&D Systems), 10 ng/ml recombinant IL-4 (eBioscience), 5 mg/ml partially rescue the GC phenotype by prolonging the survival of aCD40 (eBioscience), 10 mg/ml aIgM F(ab9)2 (Jackson Immuno- the B cells, but there remains an intrinsic block in plasma cell Research), and 10 mg/ml LPS (Sigma). To assess the effect of caspase differentiation, as well as Ab production. inhibition on proliferation, B cells were stimulated in the presence of the pan caspase inhibitor IDN-6556 at 10 mM. For cell cycle analysis, splenic B cells were stimulated with the indicated stimuli and duration, Materials and Methods and pulsed with 10 mM of BrdU (Life Technologies) during the last hour Mice of incubation prior to harvest. The cells were fixed with 70% ice-cold ethanol, treated with 2 M HCl to denature the DNA, and washed with 2/2 The Malt1 mice have been previously described (5) and were kindly 0.1 M sodium tetraborate (pH 8.5) to neutralize the acid. The cells were +/+ provided by Dr. Vishva Dixit (Genentech). Littermate controls of Malt1 then incubated with aBrdU (BD Biosciences), resuspended in a mixture +/2 2/2 or Malt1 genotypes were generated along with Malt1 mice by in- containing RNase at 100 mg/ml and 7-aminoactinomycin D (7-AAD) at terbreeding heterozygous animals. Bcl-2 transgenic mice (Em-bcl-2-22) 5 mg/ml, and analyzed by flow cytometry. For combined apoptosis and 2/2 used for breeding with Malt1 animals were purchased from The proliferation assays, eFluor670-labeled B cells were cultured as indi- Jackson Laboratory. The mice were housed in a pathogen-free environment cated and stained with Annexin-V-FITC (BioVision) according to the in the animal facility at the Sanford Burnham Prebys Medical Discovery manufacturer’s instructions. Institute. All experiments conformed to the ethical principles and guide- lines approved by the Institutional Animal Care and User Committee. Histology Bone marrow chimeras Spleens were embedded in Tissue-TEK OCT compound (Sakura Finetek) and frozen at 280˚C. Frozen tissue blocks were sectioned, mounted on 2 2 For generation of B cell specific wild type (WT) and Malt1 / mice, mMT Superfrost/Plus slides (Fisher Scientific), fixed in ice-cold acetone, and mice (The Jackson Laboratory) were sublethally irradiated (5-Gy dose) blocked with PBS with 5% FBS, plus the inhibitor E-64 at 10 mM to block 2 2 and reconstituted with bone marrow (BM) from Malt1+/+ or Malt1 / endogenous cysteine proteases for the detection of MALT1 activity. The mice. Mixed chimeras were generated as follows: mMT animals were sections were stained with the following reagents: aIgD, aCD45.1, exposed to 10-Gy of irradiation and reconstituted with a 1:1 mix of BM aCD45.2, aKi67 (clone S01A15) (eBioscience), peanut agglutinin (Vector 2 2 from CD45.1 and Malt1 / mice. BM from one to two donor mice was Labs), and the MALT1 activity probe Cy5-LVSR-AOMK (23). Imaging divided among four to six recipient animals. was acquired on a Zeiss Axio ImagerM1 microscope using the Slidebook software (Intelligent Imaging Innovations). GNU Image Manipulation Immunizations Program was used for overlaying images. Citrated SRBCs (Colorado Serum Company) were washed twice with In vitro GC B cell differentiation PBS and resuspended in PBS to a final concentration of 10% (v/v). Animals were injected i.p. with 0.2 ml of SRBC suspensions. Sera CD43-depleted naive B cells were cultured in the presence of 40LB cells were collected on day 0 and day 7 postimmunization for measuring the (24), in complete RPMI 1640 supplemented with recombinant mouse IL-4 levels of SRBC-specific IgM and IgG1 levels using a flow cytometry- (eBioscience). On day 5, the cells were harvested and analyzed by flow based method. Briefly, SRBCs were incubated with varying dilutions cytometry. The Journal of Immunology 3

Quantitative real-time PCR were also recapitulated in mMT/KO mice (Fig. 1C). Interestingly, CD43-depleted splenic B cells were rested for at least 60 min and stimulated mMT/KO mice displayed a modest but significant reduction in FO Malt12/2 with 1 mg/ml aIgM F(ab9)2 for the indicated times at 37˚C. Total RNA was B cells, which we did not observe in mice (data not isolated from B cells using Trizol (Thermo Fisher Scientific) and cDNA shown). The defect in GC reactions in mMT/KO animals also was prepared using the MMLV Reverse Transcriptase cDNA Advantage correlated with abrogated production of SRBC-specific Abs kit (Clontech), according to the manufacturers’ instructions. Quantitative (Fig. 1D). These findings indicate that B cells intrinsically require real-time PCR was performed with the SsoAdvanced SYBR Green Supermix (Bio-Rad) on a CFX384 Touch Real-Time PCR Detection sys- MALT1 for both the GC response and differentiation into MZ tem (Bio-Rad). The primers were designed with the help of PrimerBank B cells. (25) and the sequences are: Bcl2 forward 59-ATGCCTTTGTGGAACTA- 2/2 TATGGC-39, Bcl2 reverse 59-GGTATGCACCCAGAGTGATGC-39, Bcl2l1 Malt1 B cells fail to initiate GC reactions forward 59-GACAAGGAGATGCAGGTATTGG-39, Bcl2l1 reverse 59- One explanation for the lack of GCs in mMT mice reconstituted TCCCGTAGAGATCCACAAAAGT-39, Bcl2ala forward 59-GGCTGAG- 2/2 CACTACCTTCAGTA-39, Bcl2a1a reverse 59-TGGCGGTATCTATG- with Malt1 BM is a failure of B cells to induce or respond to GATTCCAC-39, Rel forward 59-AGACTGCGACCTCAATGTGG-39, Rel T cell help. To address this possibility, we adoptively transferred reverse 59-GCACGGTTGTCATAAATTGGGTT-39, Irf4 forward 59- a 1:1 mixture of WT (CD45.1) to Malt12/2 (CD45.2) splenic TCCGACAGTGGTTGATCGAC-39, Irf4 reverse 59-CCTCACGATTG- B cells into mMT recipients, followed by SRBC immunization. TAGTCCTGCTT-39, E2f3 forward 59-AAACGCGGTATGATACGTCCC- 39, E2f3 reverse 59-CCATCAGGAGACTGGCTCAG-39, Gapdh forward This model also enabled us to directly compare the competitive 2/2 59-CATGGCCTTCCGTGTTCCTA-39, Gapdh reverse 59-CCTGCTTCAC- fates of Ag-activated control versus Malt1 B cells in an CACCTTCTTGAT-39. The expression level of each target gene was normalized identical environment. The WT and Malt12/2 B cells can be ΔΔ to that of Gapdh, and the changes were calculated using the cycle threshold tracked by flow cytometry using Abs specific for CD45.1 or Downloaded from method. CD45.2, respectively (Fig. 2A). Following immunization with TD Immunoblotting Ags SRBCs, B cells derived from the CD45.1 donor accounted for more than 80% of the splenic GC compartment in the recipients Freshly isolated B cells were cultured in complete RPMI 1640 with or + + without stimuli. After 24 h, cells were collected and lysed with RIPA buffer (Fig. 2B), whereas the distribution of CD45.1 and CD45.2 (PBS, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 10 mM EDTA) plus populations within the total B cells remained equal.

10 mM sodium fluoride and complete protease inhibitor mixture (Roche). The adoptive transfer approach yielded only a small number of http://www.jimmunol.org/ The protein content of cleared lysates was measured using the BCA Protein donor-derived B cells, rendering further characterization difficult. Assay kit (Thermo Fisher Scientific). Lysates were resolved on 4–12% or 10% polyacrylamide Bis-Tris gel (Bio-Rad or Invitrogen) and transferred Therefore, we also employed a congenic mixed chimera approach onto polyvinylidene difluoride membrane (EMD Millipore). The mem- by reconstituting lethally irradiated mMT mice with a 1:1 mixture brane was probed for the indicated proteins. The following Abs were of CD45.1 to Malt12/2 BM, yielding mMT/mixed animals. His- purchased from Cell Signaling: anti-MALT1, -Bcl-2 (D17C4), -b-actin tologic analysis of spleen sections from the SRBC-immunized (13E5), -phospho-Rb (Ser807/811) (D20B12), -cyclin D3 (DCS22), -c-Myc (D84C12), -survivin (71G4B7), and -total Akt. Anti-Mcl-1 was purchased mMT/mixed chimeras was in agreement with what we have ob- from Rockland Immunochemicals, anti-cyclin D2 (M-20) from Santa Cruz, served with the adoptive transfer model, revealing a predominance and anti-Bcl-xL from BD Biosciences. of CD45.1+ cells in the GC region defined by peanut agglutinin Primary Abs were then detected with HRP-labeled donkey anti-rabbit or (PNA) positivity (Supplemental Fig. 1A, 1B). We also included by guest on September 24, 2021 anti-mouse Abs (Jackson ImmunoResearch) and developed with the the Ki67 marker to detect cycling B cells and noted that its ex- SuperSignal West Pico chemiluminescence kit (Thermo Fisher Scientific). pression mainly colocalized with the CD45.1-stained cells within Statistics the GC (Supplemental Fig. 1B). Bar graphs represent the mean values and the error bars represent the SD. In addition to its scaffolding role in promoting NF-kB activation, Statistical significance was calculated using two-tailed unpaired Student MALT1 also possesses a functional protease domain that shows t test or one-way ANOVA using GraphPad Prism (GraphPad Software). activity toward a number of substrates and fine-tunes NF-kB Only significant differences are indicated in the figures by asterisks as signaling through substrate cleavage (26). Using a newly devel- follows: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. oped probe that can selectively label active MALT1 (23), we were able to monitor MALT1 protease activity in situ in the spleens of Results the immunized mMT/mixed chimeric animals. Interestingly, cells B cell-intrinsic MALT1 function is required for the GC exhibiting MALT1 activity were mostly confined to the GC region response and did not appear in the FO mantle composed primarily of naive Although the importance of MALT1 for GC formation and sub- B cells (Fig. 2C, Supplemental Fig. 1C). In addition, MALT1 sequent Ab production is undisputed (5, 6, 15), it remains unclear activity mostly colocalized with the CD45.1+ cells, thus demon- whether the defects associated with MALT1 deficiency arise from strating the specificity of the probe (Fig. 2C). Taken together, our perturbed B cell or T cell activation, or a combination thereof. To data suggest that the B cell-intrinsic GC defect associated with resolve this issue, we generated chimeric animals by reconstitut- MALT1 deficiency cannot be compensated for even in the pres- ing sublethally irradiated B cell-deficient mMT mice, with BM ence of WT B cells that provide transactivating signals to cognate from control or Malt12/2 mice. The recipients bearing B cells that T cells. were MALT1 sufficient (mMT/WT) or deficient (mMT/knockout The reduction in GC B cells observed at the peak of the response [KO]) were immunized with SRBCs to assess whether they can could have resulted from either reduced entry of B cells into the GC mount an effective immune response. At 7 d postimmunization, or a failure of the early GC cells to properly expand and differ- the presence of GC B cells in both the spleens and Peyer’s patches entiate. To distinguish between these possibilities, we used an was assessed by flow cytometry. Similar to the defects reported for analytic approach that we had previously developed (27) involving Malt12/2 mice, mMT/KO animals exhibited a ∼7-fold reduction immunization with SRBCs, which induces a robust TD response in B220+GL7+Fas+ GC B cells (Fig. 1A). Histology of spleen with well-defined kinetics, and examination of the progression of sections from the immunized mMT/KO chimera also revealed GC B cell differentiation in the mMT/mixed chimera setting. First, almost no PNA+ GC cells in the follicles, despite the presence of PNA was used as a marker to encompass all stages of GC B cell organized CD35+ FO dendritic cell networks (Fig. 1B). The re- development. After immunization, the proportion of PNA+ B cells ductions in the total splenic B cell pool and MZ B compartment that were of Malt12/2 (CD45.2) origin was diminished (Fig. 2D, 4 B CELL-INTRINSIC MALT1 FUNCTION

FIGURE 1. B cells intrinsically require Downloaded from MALT1 for GC formation and MZ B differentiation. Sublethally irradiated mMT mice were reconstituted with Malt1+/+ (mMT/WT) or Malt12/2 (mMT/KO) BM, immunized with SRBCs, and analyzed on day 7 (n = 4 per group). (A) Representative FACS plots (left panel) and + + the frequencies of GL7 FAS GC B cells as a http://www.jimmunol.org/ percentage of total B220+ B cells (right panel) in both the spleen and Peyer’s patches are shown. (B) Immunofluorescent staining of the spleens and Peyer’s patches performed using Abs specific for B220 (APC) and CD35 (FITC), as well as PNA (PE) to detect total B cells, FO dendritic cells, and GC B cells, respectively. The images are shown at original magnification 310. Scale bar, 100 mm. (C) The frequencies of total B cells, FO B cells (CD21loCD23hi), and MZ B by guest on September 24, 2021 (CD21hiCD23) cells in the spleens were also assessed by flow cytometry. (D) Serum levels of SRBC-specific IgM and IgG1 Abs on day 0 and day 7 postimmunization were determined by flow cytometry and mean fluorescence intensities were plotted.

upper panel). We next incorporated GL7 and IgD staining to re- jority of the PNA+ B cells within the CD45.2 gate retained the solve GC B cell maturation, which proceeds from an IgD+GL72 IgD+GL72 phenotype, and ,10% of the cells matured into to an IgD2GL7+ stage (27). Focusing on the CD45.1+ population, IgD2GL7+ GC B cells (Fig. 2E). Collectively, these results sug- there was an expansion of IgD2GL7+ GC B cells, accompanied by gest that GC initiation is defective in Malt12/2 animals and that the a decrease in IgD+GL72 early GC B cells. In contrast, the ma- few PNA+IgD+GL72 early GC B cells that are present also fail to The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. B cells intrinsically require MALT1 to enter GC reaction. (A and B) CD43-depleted splenic B cells were purified from CD45.1 and Malt12/2 animals, mixed at a 1:1 ratio, and adoptively transferred into mMT mice. The mMT recipients were immunized with SRBCs 24 h later, and analyzed on day 7 postimmunization (n =5).(A) Representative FACS plots depicting the gating strategy in the recipient mice. The distribution of CD45.1 and Malt12/2 B cells in the donor cells is shown in the upper left panel. (B) Frequencies of CD45.1-derived and Malt12/2-derived cells within total B cells (B220+ gate) and GC B population (GC B gate) were plotted. (C–G) Lethally irradiated mMT mice were reconstituted with a 1:1 mix of CD45.1 and Malt12/2 BM. At 8 wk after reconstitution, the recipients (mMT/mixed) were immunized with SRBCs and analyzed on days 4, 7, or 10 (n = 3–4 per group). (C) Representative histology of the spleen cross-sections of mMT/ mixed animals on day 7 postimmunization. The GC region was outlined based on PNA staining of a consecutive section (top panel). The distribution of CD45.1- derived versus Malt12/2-derived cells was determined by staining for CD45.1 or CD45.2, respectively, and MALT1 protease activity was assessed by incubation with Cy5-LVSR-AOMK (right panel). The images are shown at original magnification 340. Scale bar, 50 mm. (D) Splenocytes were analyzed for CD45 allotypes, then the frequencies of B220+PNA+ GC B cells within the CD45.1 or CD45.2 population were determined by flow cytometry. (E) Representative FACS plots showing the progression of GCs in the B220+PNA+ populations (left panel). The frequencies of early GL72IgD+ and mature GL7+IgD2 GC B cells were plotted (right panels). (F) The frequencies of B220loCD138+ plasma cells within the CD45.1 or CD45.2 population were determined by flow cytometry. (G)CD3+CD4+ T cells were first + + analyzed for CD45.1 status and the frequencies of ICSO PD1 TFH in the spleens within the CD45.1 or CD45.2 population were determined by flow cytometry.

2/2 mature and expand. The defect in GC initiation paralleled the re- creased TFH population of Malt1 origin on day 7, suggesting 2/2 + duced frequency of Malt1 -derived B220loCD138 plasma cells that MALT1-deﬁcient T cells harbor an intrinsic block in TFH dif- on days 7 and 10 (Fig. 2F). Interestingly, we also observed a de- ferentiation (Fig. 2G). 6 B CELL-INTRINSIC MALT1 FUNCTION

Malt12/2 B cells can be induced into GC phenotype in vitro both WT and Malt12/2 B cells upon aIgM treatment (Fig. 4B). Taken together, these results suggest there is a delay rather than The inability to mount a GC response translates into a failure to 2/2 generate memory B cells and long-lived plasma cells. To determine lack of proliferative response of Malt1 B cells to BCR cross- how extensive the defect in GC formation is, we adopted an in vitro linking. The finding that the proliferation machinery remains functional culture system that has been previously described (24) to propagate 2/2 2/2 GC phenotype B cells. When naive WT B cells were cultured on in Malt1 B cells raised the possibility that most Malt1 the 40LB feeder cells, which are BALB/c 3T3 fibroblasts stably B cells undergo apoptosis before they have a chance to divide, transfected with CD40L and BAFF, they underwent massive ex- although it is unclear whether this occurs preferentially during the pansion and acquired the GL7+FAS+ GC phenotype, and are S or G2/M phase of the cell cycle. This notion prompted us to examine the viability of MALT1-deficient B cells in response to a termed induced GC B (iGB) cells (Fig. 3, left panels). Supple- 2/2 menting the culture with IL-4 also induced BCR class switching panel of stimuli. There was a reduction in viability when Malt1 from IgM to IgG1 (Fig. 3, middle panels). Strikingly, we were able B cells were cultured in media alone or stimulated with aIgM to efficiently induce the Malt12/2 B cells to differentiate into (Fig. 4C). Although the addition of BAFF or aCD40 to aIgM + + treatment was able to enhance the viability of WT B cells, it failed GL7 FAS GC-like B cells after 5 d in culture with 40LB cells. 2/2 Similar to the iGB cells derived from WT B cells, ∼50% of the to achieve the same effect on Malt1 B cells. We previously 2/2 noted that LPS- or aCD40-induced proliferation remained intact Malt1 iGB cells expressed IgG1, indicating that the class- 2/2 2/2 switching machinery in MALT1-deficient B cells remains intact. in Malt1 B cells and, consistently, Malt1 B cells survived Although a small fraction of the Malt12/2 iGB cells differentiated just as well as WT B cells when stimulated with either factor, into B220loCD138+ plasmablasts, we noted that this population suggesting that both the proliferative and survival defects are re- Downloaded from was significantly reduced relative to WT iGB cells (Fig. 3, right stricted to signaling through the BCR. Although the addition panels). Thus, Malt12/2 FO B cells proliferate normally to T cell- of BAFF failed to increase the viability of aIgM-stimulated Malt12/2 B cells compared with cells in media, BAFF treat- derived stimuli to adopt a GC B cell phenotype, but are impaired 2/2 in maturation toward the plasma cell lineage. ment alone improved the survival of Malt1 B cells. Alto- gether, these findings raise the intriguing possibility that in the 2/2

BCR-activated Malt1 B cells are less proliferative and absence of MALT1, BCR engagement induces a dominant pro- http://www.jimmunol.org/ more apoptotic apoptotic signal. The role of MALT1 in B cell proliferation in response to BCR To examine more closely the interplay of proliferation and engagement remains unclear due to conflicting results in the liter- survival, we used combined proliferation dye and Annexin-V la- ature (5, 6). We observed that FO (MZ B cell depleted) Malt12/2 beling to track cell division and apoptosis induced by aIgM 2/2 B cells proliferated robustly, comparable to WT B cells, in re- treatment. We noted that in the case of the Malt1 B cells, the + sponse to LPS or aCD40 plus IL-4, but were hyporesponsive to majority of the Annexin-V apoptotic cells did not dilute the aIgM stimuli (Supplemental Fig. 2). To investigate the nature of proliferation dye, suggesting that the cells underwent apoptosis this proliferative defect, we performed cell cycle analysis using before cell division took place (Fig. 4D). In line with a recent BrdU incorporation in combination with the DNA dye 7-AAD. report (15), the addition of IL-4, but not BAFF, was able to induce by guest on September 24, 2021 2/2 We observed a significant reduction in S-phase entry of Malt12/2 proliferation of Malt1 B cells and improve their viability. LPS B cells at 24 h after aIgM treatment. However, by 48 h post- alone was found to provide strong mitogenic signals to both WT 2/2 stimulation, the percentage of Malt12/2 B cells in the S as well as and Malt1 B cells, and we did not observe any significant G2/M phase increased to levels comparable to WT B cells (Fig. difference in proliferation and survival when LPS was adminis- 2/2 4A). Normal B cells induce expression of both cyclins D2 and D3 tered in combination with aIgM. In contrast, WT and Malt1 upon stimulation with aIgM (28). The two D-type cyclins activate B cells responded differently to combined aIgM and aCD40 the G1 kinases and target the retinoblastoma gene product for treatment. Although the addition of aCD40 was able to rescue 2/2 phosphorylation, thus freeing the E2F protein to drive transcrip- BCR-induced proliferation in Malt1 B cells to some extent, tion of genes required for the transition from the G1 to S phase. In there were fewer rounds of division compared with WT B cells line with the cell cycle analysis, we observed upregulation of stimulated under the same conditions, and there were far fewer 2/2 cyclins D2 and D3, and subsequent induction of phospho-Rb in viable Annexin-V Malt1 B cells. This result is consistent with a dominant proapoptotic effect of BCR signaling in the absence of MALT1. Next, we sought to determine if rescuing survival can overcome the impairment in proliferative response to aIgM stimulation. When treated with the broad spectrum caspase inhibitor IDN- 6556, the viability of Malt12/2 B cells in media alone or stimulated with aIgM was increased to a level comparable with that of WT B cells (Fig. 4E). Moreover, in the presence of IDN-6556, Malt12/2 B cells stimulated with aIgM exhibited a proliferative profile similar to WT B cells (Fig. 4F). Collectively, our findings indicate that the delay in cell cycle entry and the increased pro- pensity to caspase-dependent apoptosis account for the impaired proliferative response of Malt12/2 B cells to BCR stimulation. FIGURE 3. MALT1 is dispensable for in vitro GC differentiation under 2 2 strong cytokine stimulation. WT and Malt1 / splenic B cells were cultured T cell-derived help fails to rescue defects in BCR-activated with IL-4 on 40LB feeder cells for 5 d to allow for induction of phenotypically Malt12/2 B cells GC B cells. The iGB cells were characterized by flow cytometric analysis of 2/2 surface markers. Representative plots are shown. The values indicate mean To rule out the possibility that Malt1 B cells are simply anergic frequencies 6 SD from at least four independent experiments (n = 4–5 per to BCR crosslinking, the levels of various activation markers on genotype). the cell surface were assessed by flow cytometry. When stimulated The Journal of Immunology 7

with aIgM, Malt12/2 B cells upregulated CD5, CD86, MHC II, CD69, and CD80 to levels comparable with WT B cells (Fig. 5A). Notable differences were observed in the induction of CD25 and downregulation of CD62L on WT B cells and, surprisingly, enhanced basal expression of MHC II on Malt12/2 B cells. During TD immune responses, Ag-activated B cells migrate to the B–T zone boundary to seek help from cognate T cells. Accessory signals, notably CD40 and CD40L interactions, are necessary to drive the initial B cell proliferation and induction of GC formation. Proﬁling of activation markers on BCR-activated Malt12/2 B cells suggests that they retain the ability to prime T cells. Al- though we found that the combination of aIgM and aCD40 stimuli were able to partially restore aIgM-induced proliferation in Malt12/2 B cells (Fig. 4D), Malt12/2 B cells did not survive well under the combined stimulation. These ﬁndings prompted us to examine the fate of Malt12/2 B cell upon receiving T cell help. We mimicked the events in the GC response by pretreating B cells with aIgM, followed by aCD40 stimulation. It has been shown

that washing out aIgM after 24 h did not prevent the ﬁrst division, Downloaded from but most of the cells died instead of completing further divisions (29). However, subsequent engagement of CD40 was sufﬁcient to induce further proliferation of WT B cells and rescue cell death (Fig. 5B, 5C). In contrast, Malt12/2 B cells failed to proliferate and were more prone to apoptosis, verifying that CD40 signals

delivered by the T cells cannot correct the proliferation and sur- http://www.jimmunol.org/ vival defects in BCR-activated Malt12/2 B cells. Interestingly, Malt12/2 B cells primed with aCD40 were able to complete one round of cell division upon subsequent aIgM stimulation, but their viability was also markedly reduced compared with that of WT B cells. In the presence of IL-4, Malt12/2 B cells underwent robust proliferation regardless of the stimulating conditions (Fig. 5B). Thus, the impairment in both proliferative and survival response to signaling through BCR and CD40 may explain why GC initiation is defective in Malt12/2 Bcells. by guest on September 24, 2021 Expressions of Bcl-2 and Bcl-xL are reduced in Malt12/2 B cells PKCb is situated upstream of the CBM complex and controls assembly of the complex via phosphorylation of CARMA1 upon BCR engagement (30). PKCb-deﬁcient animals share many of the defects observed in Malt12/2 mice. In particular, BCR-dependent proliferation and survival in Pkcb2/2 B cells are impaired due to defective expression of Bcl-2 and Bcl-xL (31, 32). Thus, we examined the levels of various anti-apoptotic proteins in BCR-activated WT and Malt12/2 B cells. Although WT B cells upregulated Bcl-xL

were plotted. Data are representative of three independent experiments (n = 3–4 per group). (B) Immunoblot analysis of WT and Malt12/2 splenic B cells that were freshly isolated or cultured with or without aIgM for 24 h. Blots are representative of at least three experiments. (C) Viability of WT and Malt12/2 cultured with indicated stimuli for 3 d was assessed by FSC versus SSC using flow cytometry. Results from three experiments were pooled together (n = 3 per genotype). (D) WT and Malt12/2 splenic B cells were cultured in the presence of aIgM alone or in combination with other stimuli for 3 d. Cell proliferation and apoptosis were evaluated by combined eFluor670 dilution and Annexin-V staining. The cells were gated based on their Annexin-V status, and the percentage of cells within each gate is indicated. The result is representative of three independent experiments (n = 3 per genotype). (E and F) WT and Malt12/2 splenic B cells FIGURE 4. MALT1 is required for efficient cell cycle entry and pro- were cultured as indicated for 3 d with or without IDN-6556 and assessed tection from caspase-dependent apoptosis upon BCR crosslinking. (A)WT for (E) viability (FSC versus SSC profile) and (F) cell proliferation and Malt12/2 splenic B cells were stimulated as indicated for 24 or 48 h. (eFluor670 dilution) by flow cytometry. Results from two experiments The cells were pulsed with BrdU for 1 h prior to harvest, fixed and per- were pooled (n = 2–3 per genotype). Representative overlaid histograms of meabilized, followed by staining with anti-BrdU and 7-AAD. The per- eFluor670 intensities of unstimulated (gray, shaded) and stimulated (black centages of cells in S phase (top panels) and G2/M phase (bottom panels) line) B cells is shown in (F). FSC, forward light scatter; SSC, side scatter. 8 B CELL-INTRINSIC MALT1 FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. Help from T cells cannot rescue the BCR-induced survival and proliferative defects in Malt12/2 B cells. (A) Activation of Malt12/2 B cells in response to aIgM stimulation was assessed by flow cytometry. Overlaid histograms of a panel of surface markers in control (red) and Malt12/2 B cells (blue) are shown. The data are representative of three independent experiments (n = 3 per genotype). (B and C)WTorMalt12/2 splenic B cells were stimulated with aIgM or aCD40 for 24 h, washed extensively to remove the stimulus, and reactivated with aCD40 or aIgM, respectively. Cells were harvested at 24 or 48 h post second stimulus and assessed by flow cytometry analysis of combined Annexin-V staining and eFluor670 dilution. (B)FACS plots showing the proliferative versus survival profiles are representative of five experiments (n = 5–6 per genotype). (C) Viability was determined by the Annexin-V negative gate and the results from five experiments were pooled and plotted. when cultured in the presence of aIgM alone or in combination with exception of Bcl2l1, the profiles of all the other genes in response to BAFF or aCD40, Bcl-xL expression was reduced in Malt12/2 B cells aIgM stimulation were similar between WT and Malt12/2 B cells after stimulation (Fig. 6A). Intriguingly, whereas Bcl-2 is abundant in (Fig. 6B). Thus, the reduction in Bcl-2 is likely regulated at the level unstimulated B cells there was a reduction in Bcl-2 expression in of protein stability. These data suggest that the impaired survival in Malt12/2 B cells upon stimulation. In contrast, the Mcl-1 expression BCR-activated Malt12/2 B cells is due to reduced transcription of was comparable between WT and Malt12/2 B cells. Malt12/2 B cells Bcl2l1 and impaired stability of Bcl-2. also induced c-Myc expression upon stimulation, indicating that they Enforced Bcl-2 expression partially rescues GC impairment in were responsive to stimulation. Bcl2l1 is a direct target of the tran- 2/2 scription factor c-Rel (33), which was found to be selectively acti- Malt1 mice vatedbyMALT1(8).Thisprompted us to monitor the transcript Given that treatment with a caspase inhibitor was able to restore levels of several known c-Rel target genes in WT versus Malt12/2 proliferation and survival of BCR-activated Malt12/2 B cells, we B cells after aIgM treatment by time-course real-time PCR. With the sought to determine whether ectopic expression of Bcl-2 could The Journal of Immunology 9

BM from Bcl-2 Tg 3 Malt1+/2 (mMT/Bcl-2 Tg 3 Malt1+/2)or Bcl-2 Tg 3 Malt12/2 (mMT/Bcl-2 Tg 3 Malt12/2) littermates into sublethally irradiated mMT mice. Remarkably, immunization with SRBC elicited GC responses in mMT mice reconstituted with Bcl-2 Tg 3 Malt12/2 BM, albeit not as robust as in mMT/Bcl-2 Tg 3 Malt1+/2 (Fig. 7A, top panels). The mMT/Bcl-2 Tg 3 Malt12/2 recipients reconstituted 34% of the GL7+FAS+ cells as a proportion of total B220+ B cells relative to mMT/Bcl-2 Tg 3 Malt1+/2 mice. A similar proportion of the GC B cells in both groups stained positive for IgG1 (Fig. 7A, middle panels), suggesting that MALT1 is not essential for class-switch recombination. However, the frequency of plasma cells was drastically reduced in mMT/ Bcl-2 Tg 3 Malt12/2 animals (Fig. 7A, lower panels). In addition, we found that forced Bcl-2 expression did not rescue the defect in MZ B cell differentiation observed in Malt12/2 animals (Fig. 7B). Although the two groups showed comparable frequencies of splenic CD4+ T cells, mMT/Bcl-2 Tg 3 Malt12/2 animals exhibited a 2-fold reduction in TFH population (Fig. 7C), which

correlated with the reduction in GC B cells. Downloaded from To investigate whether the reduction in GC B cells observed at the peak of GC response was due to impaired maturation of early GC founder cells, PNA, GL7, and IgD staining was incorporated to resolve subsets of GC B cells in the immunized mMT chimeras. The entire GC compartment was ﬁrst identiﬁed by staining with

the pan-GC marker PNA, which showed a 3-fold reduction in http://www.jimmunol.org/ mMT/Bcl-2 Tg 3 Malt12/2 animals (Fig. 7D, upper panels). However, there was no appreciable difference in the proportions of mature IgD2GL7+ and early IgD+GL72 GC B cells in mMT/Bcl-2 Tg 3 Malt12/2 relative to mMT/Bcl-2 Tg 3 Malt1+/2 mice (Fig. 7D, middle and bottom panels). When histology was performed on mMT/Bcl-2 Tg 3 Malt12/2 spleen cross-sections, we were able to detect PNA+ GC clusters in all the follicles, but the size of the GC clusters was smaller compared with those seen in the mMT/Bcl-2 Tg 3 Malt1+/2 animals (Fig. 7E). The effect on by guest on September 24, 2021 the Ab response was also assessed, and as expected, SRBC immunization led to the production of SRBC-specific IgM and IgG Abs in mMT/Bcl-2 Tg 3 Malt1+/2 animals. Consistent with the block in plasma cell differentiation, the levels of SRBC-specific Abs were severely diminished in immunized mMT/Bcl-2 Tg 3 Malt12/2 chimeras (Fig. 7F). Taken together, in the presence of WT T cells, forced expression of Bcl-2 in B cells partially restored GC formation in MALT1-deficient animals, but failed to rescue plasma cell differentiation or MZ B cell formation. Bcl2 transgene does not correct proliferative defects in Malt12/2 B cells To investigate whether the Bcl2 transgene rescues the response to BCR stimulation, proliferation and apoptosis of splenic B cells FIGURE 6. MALT1 is required for sustained Bcl-2 expression and Bcl- were monitored by combined Annexin-V and eFluor670 labeling. xL upregulation in BCR-activated B cells. (A) Immunoblot analysis of WT +/2 2 2 Similar to WT B cells, Bcl-2 Tg 3 Malt1 B cells underwent and Malt1 / splenic B cells stimulated as indicated for 24 h. Blots are representative of three independent experiments (n = 3 per genotype). (B) several rounds of division after 72 h in culture in response to BCR Time-course induction of several c-Rel target genes in response to aIgM triggering, and proliferation was further enhanced by the addition 2/2 treatment was monitored by real-time PCR. WT (n = 2, black) and Malt12/2 or BAFF or aCD40 (Fig. 8A). Notably, Bcl-2 Tg 3 Malt1

(n = 3, red) splenic B cells were stimulated with 1 mgofaIgM F(ab9)2 for 4, B cells cycled at most two rounds to BCR ligation, although vi- 8, 12, and 24 h. At each time point, the expression of each gene was first ability was increased ∼6-fold by ectopic Bcl-2 expression com- corrected for expression of the reference gene Gapdh, then the fold-changes, pared with Malt12/2 B cells (see Fig. 4D). Although the addition relative to the zero-time point of the same animal, were plotted. (C)Ex- of BAFF or aCD40 had a minimal effect on aIgM-induced pro- 2/2 pression of same set of c-Rel target genes at 4 h poststimulation in Malt1 liferation in Bcl-2 Tg 3 Malt12/2 B cells, the inclusion of either BcellswasnormalizedtothatofWT. IL-4 or LPS triggered a robust proliferative profile that was similar to what Bcl-2 Tg 3 Malt1+/2 B cells exhibited. The survival of rescue MZ B cell development and TD responses in Malt12/2 Bcl-2 Tg 3 Malt1+/2 and Bcl-2 Tg 3 Malt12/2 B cells in re- mice. Malt12/2 mice were interbred with Em-Bcl-2-22 transgenic sponse to other stimuli was also explored, and the incubation mice, which specifically express the human Bcl2 transgene in the period was extended from 72 to 96 h to determine if there was a B lineage (34), and chimeric mice were generated by transferring greater differential. Viability was similar for Bcl-2 Tg 3 Malt1+/2 10 B CELL-INTRINSIC MALT1 FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. Bcl2 transgene partially restored TD Ag-induced GC formation but failed to rescue the block in plasma cell differentiation in Malt12/2 B cells. Sublethally irradiated (5 Gy) mMT mice were reconstituted with BM from Bcl-2 -Tg 3 Malt1+/2 or Bcl-2 Tg 3 Malt12/2 (two donors from each genotype into four recipients), immunized with SRBCs, and analyzed on day 7 postimmunization. (A) Total B220+ B cells, GC B cells (B220+GL7+Fas+), IgG1+ class-switched GC B cells, and plasma cells (B220loCD138+) in the spleens were assessed by flow cytometry. (B)FOB (Figure legend continues) The Journal of Immunology 11 and Bcl-2 Tg 3 Malt12/2 B cells when cultured under resting Further studies are also needed to elucidate the mechanism of how conditions or in the presence of stimuli other than aIgM (Fig. 8B). MALT1 protease activity is regulated during the GC response and In contrast, despite the enforced Bcl-2 expression, Bcl-2 Tg 3 the in vivo consequences of MALT1 substrate cleavage. In this Malt12/2 B cells exhibited more cell death when stimulated with sense, it would be interesting to determine whether MALT1 protease aIgM, and the addition of BAFF or aCD40 failed to enhance activity mirrors that of NF-kB signaling. viability. Lastly, we examined the differentiation of plasma cells Importantly, our data demonstrate that the requirement for under defined in vitro conditions to rule out other B cell extrinsic MALT1 is mainly restricted to BCR-induced signaling. Divergent variables. In support of our in vivo findings, the generation of views on the involvement of MALT1 in mitogen-induced prolif- B220loCD138+ Ab-secreting cells was significantly reduced in eration exist since the first Malt12/2 mice were described. Bcl-2 Tg 3 Malt12/2 and Malt12/2 B cells after 3 d of culture Whereas Ruefli-Brasse et al. (5) reported that proliferative re- with LPS or aCD40+IL-4, compared with their respective litter- sponses to aIgM, aCD40, or LPS were all impaired in Malt12/2 mate controls (Fig. 8C, 8D). Our collective results thus suggest B cells, Ruland et al. (6) argued that MALT1 is dispensable for that MALT1 is selectively required for the survival of resting and proliferation induced by any of the three stimuli. Our results, using BCR-activated B cells, and that forced expression of Bcl-2 can eFluor670 dye dilution to directly monitor cell division instead partially overcome this proapoptotic effect but it does not enable of 3H-thymidine incorporation to measure DNA replication, are the differentiation of Ab-secreting cells. intermediate and more in line with observations made by Ferch et al. (8). Also consistent with recent findings reported by Bornancin Discussion et al. (15), we showed that costimulation of IL-4 rectified both the 2 2 MALT1 has been recognized as an essential component of the Ag proliferative and survival defects in BCR-activated Malt1 / Downloaded from receptor signaling pathway, regulating NF-kB activity via both B cells, most likely via induction of the STAT6 pathway (47). A scaffolding (35) and protease functions (36–39). However, its similar defect in BCR-induced proliferation was seen in mice specific role in B cells has not been fully characterized. In this deficient in CARMA-1, BCL-10, Btk, and PKCb, thus supporting study, we provide several lines of evidence for a cell-intrinsic role the requirement of the CBM complex downstream of the BCR-Btk of MALT1 in regulating B cell immune function. Using chimeric axis (43, 48–50). Although CD40 signaling remains intact in 2/2 mouse models, we demonstrated that there is a B cell-intrinsic Malt1 B cells, when aCD40 treatment was provided at the http://www.jimmunol.org/ requirement for MALT1 in the GC response to TD Ags. Central same time, or preceding BCR stimulation, it had a minimal effect to BCR-induced NF-kB signaling is Bruton’s tyrosine kinase on survival and only partially rescued proliferation. Costimulation (Btk), which is primarily responsible for the activation of PLCg2 with BAFF also shared a similar proliferation/survival profile as and subsequently PKCb, leading to the assembly of the CBM stimulation of BCR alone, suggesting that the defect in BCR- complex. Mutation or inhibition of Btk has been shown to result in induced signaling in the absence of MALT1 is dominant over a compromised GC response (40, 41). In accordance with the BAFF-induced signaling. BCR-Btk pathway, deficiency in any of the components of the Our results indicate that reduced viability of BCR-activated CBM complex led to impaired TD humoral response (5, 6, 31, 42– Malt12/2 B cells is due to reduced expression of Bcl-2 and lack 44). It is thought that NF-kB is dispensable for GC B cell pro- of Bcl-xL upregulation, on both mRNA and protein levels. Inter- by guest on September 24, 2021 liferation, because GC B cells fail to express most NF-kB target estingly, there was no difference in viability between Bcl-2 Tg 3 genes and NF-kB pathway components (45). This notion is sup- Malt12/2 and Bcl-2 Tg 3 Malt1+/2 B cells that were left in media ported by a later study showing that BCR signaling is dampened in alone, but viability of Bcl-2 Tg 3 Malt12/2 B cells was reduced in GC B cells, due to phosphatase activity, and increases again in the all stimulating conditions involving aIgM stimulation, suggesting light zone (46). A recent study dissecting the differential roles of that the protective mechanism of MALT1 following BCR activa- the NF-kB subunits RelA and c-Rel in the GC response also tion also includes a Bcl-2 independent component. Although revealed that although c-Rel is required for GC maintenance, overexpression of either Bcl-2 or Bcl-xL leads to accumulation of activation of RelA is indispensable at later stages for the gener- mature B cells, Bcl-xL but not Bcl-2 becomes rapidly upregulated ation of plasma cells (14). Thus, we postulate that BCR-induced upon activation via surface Ig, LPS, or CD40 stimulation (51, 52). NF-kB induction is more associated with GC initiation as a con- Furthermore, within the GC, CD40-CD40L interactions rescue sequence of Ag activation. Consistently, our results indicate that centrocytes from apoptosis primarily via the induction of Bcl-xL the block in Malt12/2 animals occurs at the early stage of the GC (53), which can also be mimicked by the ectopic expression of reaction before proliferative expansion takes place. Interestingly, Bcl-2 (54). More definitive experiments are required to verify using an activity-based probe, we showed that MALT1 proteolytic whether Bcl-xL expression in B cells can completely reverse the activity can only be detected within the GC region. A recent report defects associated with MALT1 deficiency. How MALT1 reg- on MALT1 protease-inactive mice revealed that although PMA- ulates the expression of both Bcl-2 and Bcl-xL also awaits fur- induced NF-kB activation remains intact in B cells, the lack of ther analysis. Previous studies have identified c-Rel as a MALT1 proteolytic activity partially affected the GC response protector of B cells from BCR-mediated apoptosis, partly via (15). Whether MALT1-mediated cleavage events are necessary for induction of Bcl-xL and A1 (13, 33). In contrast, the link be- the maintenance of GC reactions and GC exit would require the tween Bcl-2 expression and NF-kB activation in B cells is not characterization of additional inducible deletion mouse models. firmly established.

+ + cells (CD21loCD23hi) and MZ B cells (CD21hiCD23lo) in the spleens were assessed by flow cytometry. (C) Total CD3 CD4 T pool and TFH differentiation, indicated by PD-1 and ICOS staining, were determined by flow cytometry. The values represent mean frequencies 6 SD within the indicated gates of four animals per group. (D) Total GC B cells in the immunized recipients were first identified by B220+PNA+ staining (upper panels) and were further characterized for GC maturation by their GL7 and IgD profile. A representative FACS profile from each group is shown (middle panels) and the frequencies in the IgD2GL7+ and IgD+GL72 gates were plotted (lower panels). (E) Representative images of spleen sections stained with a-IgD (PE) and PNA (FITC). The images were taken at original magnification 35. (F) Levels of SRBC-specific IgM and IgG1 Abs in the serum collected on day 0 and day 7 postimmunization were measured by flow cytometry. The graphs show mean fluorescence intensities. 12 B CELL-INTRINSIC MALT1 FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 8. MALT1 regulates survival and proliferation in BCR-stimulated B cells. (A) CD43-depleted splenic B cells from Bcl-2 Tg 3 Malt1+/2 or Bcl- 2Tg3 Malt12/2 mice were cultured with the indicated stimuli for 3 d. Cell proliferation and apoptosis were evaluated by combined eFluor670 dilution and Annexin-V staining. (B) Histogram showing the viability of Bcl-2 Tg 3 Malt1+/2 (n = 2), Bcl-2 Tg 3 Malt12/2 (n = 3), Malt1+/2 (n = 1), and Malt12/2 (n = 1) B cells cultured as indicated for 4 d. The percentage of cells in the live gate according to the FSC versus SSC profile was determined by flow cytometry. Error bars indicate SD. (C and D) Purified B cells cultured with the indicated mitogens for 4 d were analyzed by flow cytometry to assess the formation of Ab-secreting cells, identified by their B220loCD138+ phenotype. The data are representative of at least four independent experiments (n = 4–5 per genotype). FSC, forward light scatter; SSC, side scatter.

Our results indicate that the Bcl2 transgene, together with the to successfully prime TFH differentiation. It has been reported that presence of WT T cells, led to a partial rescue of GC formation by sustained Ag presentation by dendritic cells facilitates early TFH prolonging the survival of MALT1-deficient B cells. Our mMT/ formation in the absence of B cells (58). These findings suggest that mixed chimera model showed that even in the same environment the block in TFH differentiation may also arise from a cell-intrinsic as WT T cells, MALT1-deficient T cells less readily differentiate defect in Malt12/2 T cells. The MALT1 substrate Roquin is a likely into the TFH phenotype. TFH cells have been acknowledged as the candidate because it inhibits ICOS, and the loss of both Roquin-1 key cell type required for GC formation and the generation of and Roquin-2 has been reported to result in spontaneous TFH dif- memory B cells (55). Although TFH cells provide help to Ag- ferentiation and GC development (59, 60). activated B cells, reciprocal signals from B cells are also impor- Nevertheless, there remains a B cell-intrinsic requirement for tant for the maintenance of TFH differentiation, leading to the MALT1 for the GC response, plasma cell differentiation, and Ig induction of the master regulator transcription factor Bcl-6 in either production. The partial decline in GC B cells observed in the mMT/ 2/2 cell type (56, 57). Consequently, the reduction in TFH phenotype in Bcl-2 3 Malt1 animals can be explained by the intrinsic block Malt12/2 mice may reflect the inability of MALT1-deficient B cells in BCR-induced proliferation associated with MALT1 deficiency, The Journal of Immunology 13 thereby restricting the expansion of centroblasts during early GC or in parallel pathways, such as CARMA1 or MYD88, respec- development. Although defective c-Rel translocation has been tively, because they are unlikely to respond to inhibition of Syk, implicated in Malt12/2 B cells (8), we cannot completely rule Btk, or PKCb. out the involvement of MALT1 in regulating activation of RelA as well, provided that RelA is essential for the generation of GC- Disclosures derived plasma cells (14). Our results indicate that MALT1 is The authors have no financial conflicts of interest. also indispensable for plasmablast differentiation induced by BCR-independent stimuli. The fact that LPS- and aCD40+IL-4– induced proliferation remains intact in Malt12/2 Bcellsseems References paradoxical, but our analysis of in vitro B cell culture revealed an 1. Vallabhapurapu, S., and M. Karin. 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27: 693–733. impairment in plasmablast generation. Given that differentiation 2. Thome, M., J. E. Charton, C. Pelzer, and S. Hailfinger. 2010. 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Supplemental Figure 1. Immunofluorescent staining of spleen sections in µMT/mixed chimeras.

(A) Consecutive spleen sections from SRBC-immunized µMT/mixed chimeras were first stained with PNA to visualize the GC region, which is outlined by the dashed line. (B)

The distributions of WT- and Malt1-/--derived cells within the GC were determined by staining with antibodies specific for CD45.1 and CD45.2, respectively. Proliferating cells were identified by Ki67+ staining. The images were taken at 10x magnification. Scale bar represents 50 µm. (C) Spleen sections from SRBC-immunized WT and Malt1-/- animals, as well as unimmunized WT control animal, were stained with anti-B220 antibody and PNA. MALT1 protease activity was probed with Cy5-LVSR-AOMK. The images are shown at 40x magnification and the scale bar represents 50 µM.

Supplemental Figure 2. MALT1 is selectively required for BCR-induced proliferation.

(A) Splenic FO B cells were enriched from WT and Malt1-/- mice by depletion of non-B cells (CD43+) and MZ B cells (CD9+). Purity was determined by flow cytometry. (B)

Sorted splenic FO B cells were cultured with the indicated stimuli for 3 days and cell proliferation was measured by dilution of the eFluor670 dye. The percentage of divided cells is shown in each dot plot. Representative plots from 2 independent experiments (n =

2 per genotype) are shown.